High concentrations of glutamate induce neuronal cell death (excitotoxicity), a process that contributes to cell loss in many neurological diseases. In a study aiming to elucidate the molecular mechanism underlying excitotoxicity, we established Akt/PKB deactivation as a causal mediator of neuronal death. Recently, we discovered that a small intracellular molecule, lnsP7 (diphosphoinositol-pentakisphosphate), competes with Ptdlns(3,4,5)P3 for binding to the PH domain of Akt, and thereby attenuates Akt membrane translocation and subsequent activation. This intriguing result leads us to hypothesize that lnsP7, by deactivating Akt, mediates excitotoxicity. Consistent with this idea, our preliminary data demonstrated that 1 of the 3 InsPS kinases generating lnsP7, lnsP6K2, is required for glutamate-excitotoxicity. To further test our hypothesis that Akt deactivation is mediated by direct binding of lnsP7 to Akt, association of lnsP7 with Akt in intact neurons will be examined (Aim I-A). Moreover, to evaluate to what extent lnsP7 contributes to the Akt deactivation and neuronal death, Ptdlns(3,4,5)P3 level as well as PI3K and PTEN activity will be measured (Aim I-B). To prove lnsP7 functions between Ptdlns(3,4,5)P3 and Akt as hypothesized in our model, we will examine whether elevating lnsP7 level can attenuate Akt activity in cells expressing constitutively active PI3K or Akt (Aim I-C). In addition, to determine the involvement of lnsP6K1 and lnsP6K3 in excitotoxicity, we will deplete them using siRNA technique and examine whether their depletion can suppress neuronal death (Aim II). Finally, the contribution of lnsP7 to excitotoxicity in animal brain will be investigated in a stroke model using lnsP6K2-null mice (Aim III). Results from these studies will provide new insights into the molecular mechanism of glutamate-excitotoxicity and further solidify lnsP7 and related pathways as legitimate therapeutic targets for preventing excitotoxicity-elicited neuronal death. Thus, novel strategies for drug design and therapeutic management could be created to treat a variety of neurological diseases.